fmicb-10-00510 March 22, 2019 Time: 20:29 # 1

ORIGINAL RESEARCHpublished: 22 March 2019

doi: 10.3389/fmicb.2019.00510

Edited by:Sébastien Duperron,

Muséum National d’Histoire Naturelle,France

Reviewed by:Isabelle Florent,

Muséum National d’Histoire Naturelle,France

Marie-Agnès Travers,Institut Français de Recherche pourl’Exploitation de la Mer (IFREMER),

France

*Correspondence:Letizia Modeo

letizia.modeo@unipi.it

†These authors have contributedequally to this work

‡Present address:Carolina Chiellini,

Department of Agriculture, Food,and Environment, University of Pisa,

Pisa, Italy

Specialty section:This article was submitted to

Microbial Symbioses,a section of the journal

Frontiers in Microbiology

Received: 14 September 2018Accepted: 27 February 2019

Published: 22 March 2019

Citation:Chiellini C, Pasqualetti C,

Lanzoni O, Fagorzi C, Bazzocchi C,Fani R, Petroni G and Modeo L (2019)

Harmful Effect of Rheinheimera sp.EpRS3 (Gammaproteobacteria)

Against the Protist Euplotesaediculatus (Ciliophora, Spirotrichea):

Insights Into the Ecological Roleof Antimicrobial Compounds From

Environmental Bacterial Strains.Front. Microbiol. 10:510.

doi: 10.3389/fmicb.2019.00510

Harmful Effect of Rheinheimera sp.EpRS3 (Gammaproteobacteria)Against the Protist Euplotesaediculatus (Ciliophora,Spirotrichea): Insights Into theEcological Role of AntimicrobialCompounds From EnvironmentalBacterial StrainsCarolina Chiellini1†‡, Chiara Pasqualetti2†, Olivia Lanzoni2, Camilla Fagorzi1,Chiara Bazzocchi3, Renato Fani1, Giulio Petroni2 and Letizia Modeo2*

1 Department of Biology, University of Florence, Florence, Italy, 2 Department of Biology, University of Pisa, Pisa, Italy,3 Department of Veterinary Medicine, University of Milan, Milan, Italy

Rheinheimera sp. strain EpRS3, isolated from the rhizosphere of Echinacea purpurea,is already known for its ability to produce antibacterial compounds. By use of cultureexperiments, we verified and demonstrated its harmful effect against the ciliated protistEuplotes aediculatus (strain EASCc1), which by FISH experiments resulted to harborin its cytoplasm the obligate bacterial endosymbiont Polynucleobacter necessarius(Betaproteobacteria) and the secondary endosymbiont “Candidatus Nebulobacteryamunensis” (Gammaproteobacteria). In culture experiments, the number of ciliatestreated both with liquid broth bacteria-free (Supernatant treatment) and bacteriaplus medium (Tq treatment), decreases with respect to control cells, with completedisappearance of ciliates within 6 h after Tq treatment. Results suggest thatRheinheimera sp. EpRS3 produces and releases in liquid culture one or more bioactivemolecules affecting E. aediculatus survival. TEM analysis of control (not treated) ciliatesallowed to morphologically characterize both kind of E. aediculatus endosymbionts.In treated ciliates, collected soon after the arising of cell suffering leading to death,TEM observations revealed some ultrastructural damages, indicating that P. necessariusendosymbionts went into degradation and vacuolization after both Supernatant and Tqtreatments. Additionally, TEM investigation showed that when the ciliate culture wasinoculated with Tq treatment, both a notable decrease of P. necessarius number and anincrease of damaged and degraded mitochondria occur. FISH experiments performedon treated ciliates confirmed TEM results and, by means of the specific probe hereindesigned, disclosed the presence of Rheinheimera sp. EpRS3 both inside phagosomesand free in cytoplasm in ciliates after Tq treatment. This finding suggests a putativeability of Rheinheimera sp. EpRS3 to reintroduce itself in the environment avoidingciliate digestion.

Keywords: Rheinheimera sp. EpRS3, ciliate, TEM, FISH, culture experiment, endosymbiont, antimicrobial

Frontiers in Microbiology | www.frontiersin.org 1 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 2

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

INTRODUCTION

Rheinheimera sp. strain EpRS3 is a Gammaproteobacteriumbelonging to the family Chromatiaceae. The strain was isolatedfrom the rhizospheric soil of Echinacea purpurea (Chielliniet al., 2014), a medicinal plant previously described for itsantimicrobial activity (Hudson, 2012). Rheinheimera EpRS3shows resistance to several antibiotic compounds (Mengoniet al., 2014) and can inhibit the growth of various bacteriaisolated from both E. purpurea rhizospheric soil and planttissues (Maida et al., 2016). Additionally, EpRS3 can inhibit thegrowth of opportunistic human pathogenic bacteria belonging tothe Burkholderia cepacia complex (Bcc) (Chiellini et al., 2017),as well as the growth of different bacterial pathogens (e.g.,Acinetobacter baumanni, Klebsiella pneumoniae) the majorityof which exhibit a multi-drug-resistance phenotype (Prestaet al., 2017). It has been hypothesized that the productionof antimicrobial compounds, and the subsequent antagonisticactivity of some bacterial strains belonging to the same ordifferent taxa, might play a (key) role in driving the structuringof microbial communities interacting with eukaryotic macro-organisms (Maida et al., 2016). The toxicity of bacteria belongingto Rheinheimera genus against other organisms has been alsodescribed in other strains such as GR5, showing antimicrobialactivity against Gram-positive and Gram-negative bacteria, yeast,and algae (Chlorophyceae, Trebouxiophyceae) (Chen et al.,2010; Romanenko et al., 2015). Interestingly, Rheinheimera sp.strain GR5 can synthesize an l-lysine oxidase with antimicrobialactivity associated with generation of hydrogen peroxide(Chen et al., 2010). Recently, the antibacterial toxicity of themarine bacterium Rheinheimera japonica KMM 9513 has beendescribed as well. This strain is able to exert the antimicrobialactivities against Bacillus subtilis and/or Enterococcus faeciumand Staphylococcus aureus by synthesizing phthalates anddiketopiperazines (Kalinovskaya et al., 2017). Moreover, theanti-quorum sensing activity of the diketopiperazine factorcyclo (Trp-Ser) produced by Rheinheimera aquimaris QSI02against Chromobacterium violaceum CV026 and Pseudomonasaeruginosa PA01 has been evidenced, focusing the attentionon the potentiality of this bacterial genus to interact withother bacterial species in environmental microbial communities(Sun et al., 2016).

Under an ecological point of view, the production ofbactericidal compounds from environmental bacterial strainscan be related to different factors such as the competition fornutrients and the occupation of a specific niche by eliminatingprior residents (Hibbing et al., 2010). On the other side, bacteriain the environment can produce molecules affecting the survivalof eukaryotic organisms such as fungi. This is the case forexample of some rhizospheric bacteria producing moleculeswith antifungal activity to protect the plant against pathogens(e.g., Pandey et al., 1997; Ligon et al., 2000; Cho et al., 2007).Interestingly, few studies focused the attention on bacterialantimicrobial compounds affecting the growth and survival ofprotists. An example is that of the environmental pathogenicP. aeruginosa, whose type three secretion system (TTSS) effectorproteins allow the bacterium inducing necrosis and apoptosis,

and killing of free-living Acanthamoeba species (Abd et al., 2008).Lovejoy et al. (1998) documented how Pseudoalteromonas sp.bacteria affect dinoflagellates survival, causing rapid cell lysis,and death of gymnodinoids (Gymnodinium catenatum) andraphidophytes (Chattonella marina and Heterosigma akashiwo).A deleterious effect of freshwater bacteria Janthinobacteriumlividum and C. violaceum against flagellated protists Ochromonassp. and Spumella sp. (Chrysophyceae) has also been described,by means of violacein production from bacterial strains (Matzet al., 2004b). In this scenario, a possible biocidal effect ofRheinheimera sp. EpRS3 against eukaryotic cells has becomean intriguing topic. Could EpRS3 interact with eukaryoticunicellular microorganisms in complex microbial communities(e.g., biofilm)? Could this possible interaction determine thestructuring of such communities in natural environments? Toaddress these interesting questions, we performed experimentsinvolving the ciliated protozoan Euplotes aediculatus (Ciliophora,Spirotrichea) monoclonal strain EASCc1, as a model organism tobe tested in culture experiments with Rheinheimera sp. EpRS3.

The choice of E. aediculatus as model organism is based ondifferent reasons such as the numerous previously publishedstudies, providing a wide knowledge on the biologicalcharacteristics of this relatively easily cultivable ciliate (e.g.,Sogin et al., 1986; Heckmann and Schmidt, 1987; Boscaroet al., 2012, 2013c; Vannini et al., 2017). Additionally, assymbioses involving ciliate hosts are widespread (e.g., seeVannini et al., 2010; Modeo et al., 2013b; Schrallhammeret al., 2013; Fokin et al., 2014, 2019; Castelli et al., 2018a;Lanzoni et al., 2019), fresh- and brackish-water strainsof E. aediculatus are known to host in the cytoplasm theobligate bacterial endosymbiont Polynucleobacter necessarius(Heckmann and Schmidt, 1987; Vannini et al., 2005, 2007)and, possibly, also secondary symbionts (Boscaro et al.,2012), and the strain EASCc1 used for the present studymade no exception. Indeed, from a preliminary fluorescencein situ hybridization (FISH) screening, E. aediculatus strainEASCc1 was known to host in its cytoplasm two bacterialsymbionts: P. necessarius (Betaproteobacteria) as primaryendosymbiont, and an unidentified Gammaproteobacterium assecondary endosymbiont.

The symbiotic relationship between the endosymbiont P.necessarius and its host E. aediculatus falls within obligatemutualism: the two involved species live in close proximity andinterdependently with one another so that one cannot survivewithout the other. The symbionts had assumed an obligate role:after their removal by ciliate ampicillin treatment, ciliates cannotproperly divide anymore and after one week they eventually die(Heckmann and Schmidt, 1987). Thus, the present study wasconceived to verify by means of culture experiments, and FISHand TEM analyses:

(1) The possible deleterious effect of Rheinheimera sp. EpRS3on this ciliate, taken as a model system among freshwaterubiquitous grazing protozoans, in case the deleteriouseffect occurred in few hours [i.e., Rheinheimera sp. EpRS3bioactive molecule(s) also affect eukaryotic cells]; this effecthas been herein defined as “direct” effect.

Frontiers in Microbiology | www.frontiersin.org 2 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 3

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

(2) The possible deleterious effect of Rheinheimera sp. EpRS3on this ciliate, in case the deleterious effect occurredafter about 1 day as a consequence of the depletionof the indispensable endosymbiont P. necessarius [i.e.,Rheinheimera sp. EpRS3 bioactive molecule(s) only affectbacterial cells]; this effect has been herein defined as “notdirect” effect.

(3) The possible resistance of P. necessarius to the presenceof Rheinheimera sp. EpRS3 in case E. aediculatus was notaffected at all (a kind of host protection effect like theone documented for Acanthamoeba and Legionella: Linder,1999) [i.e., Rheinheimera sp. EpRS3 bioactive molecule(s)are shadowed by eukaryotic cells and do not affect hostbacterial endosymbionts].

Both FISH and TEM investigation supported a directdeleterious effect of Rheinheimera sp. EpRS3 on Euplotescells, with mitochondria being a probable target of thebioactive molecule(s). Interestingly, TEM analysis on affectedE. aediculatus cells showed the presence of Rheinheimerasp. EpRS3 in Euplotes cytoplasm both inside and outsidedigestive vacuoles. A deleterious effect was also observed on theendosymbiont P. necessarius; the secondary endosymbiont,herein morphologically and molecularly characterized,appeared not affected.

MATERIALS AND METHODS

Euplotes aediculatus Strain EASCc1Isolation and Laboratory MaintenanceEuplotes aediculatus specimens were collected from the anaerobicside tank (0 ppt salinity) of the San Colombano activatedsludge plant placed in Lastra a Signa (Florence, Italy), inNovember 2015. A single cell was isolated from the originalpopulation and was washed several times in sterile San Benedettowater (San Benedetto S. p. A. Italy) in order to minimizethe presence of contaminating microorganisms. The cell wasthen accommodated in San Benedetto water and daily fed for5 days with a drop of cerophyll medium (CM) inoculatedwith Raoultella planticola DSM3069 (Gammaproteobacteria,Enterobacteriaceae) to induce its exponential growth. CM is acommercially available mixture of pulverized wheat grasses. Oneliter of CM is composed by Cerophyll buffer (79,75 g/l Na2HPO4and 27,1 g/l NaH2PO4), balanced salt solution (BSS = 20.8 g/lNaCl, 8 g/l MgSO4 × 7 H2O, 17 g/l MgCl2 × 6 H2O, 2,7 g/lCaCl2 × 2 H2O, and 4,6 g/l KCl), and stigmasterol (5.0 mg/mlin 99.8% ethanol). Food bacteria were stored at −20◦C in 1 mlaliquots with 50% glycerin (v/v). Each aliquot was prepared inorder to be sufficient to guarantee a good density of bacteria in1 L of CM after 24 h of incubation at a temperature of 37◦C.For the inoculation of CM, R. planticola aliquots were thawedand centrifuged for 5 min at 5,500 × g. After discharging thesupernatant, the pellet was washed and re-suspended with 1 ml ofCM. The bacteria were centrifuged again for 3 min at 5,500 × gto form a pellet. Lastly, they were resuspended in 1 ml of CM andthen inoculated in 1 L of CM. Once E. aediculatus monoclonal

strain EASCc1 was established, the culture was maintained inincubator at a temperature of 19± 1◦C, with a 12:12 h irradianceof 200 µmol photons m−2s−1 (see Boscaro et al., 2013b andCastelli et al., 2018b for details). The obtained monoclonal culturewas fed once a week with 1/4 bacterized CM of the total culturevolume. In this culture conditions, ciliate doubling time wasabout 36 h. However, all ciliates used in the experiments werekept in starving conditions for 4 days before the experimentto ensure food digestion. The culture was constantly checkedto ensure cell healthy conditions during the whole time of theexperiments. E. aediculatus cell density was calculated undera stereo-microscope (Leica Wild M3C) using three technicalreplicates of 100 µl each; the cell number was estimated by mean,and calculated for 1 ml of culture (±150 cells/ml).

Live Cell Imaging and CiliateIdentificationLiving cells were immobilized on a slide for observation withthe help of a coverslip without deforming them (Skovorodkin,1990). Living cells were examined under differential interferencecontrast (DIC) using an Orthoplan Leitz microscope (Leitz,Germany) at×300–1.250 magnifications, and photographed witha digital camera Canon S45 and True Chrome HDII. Ciliateidentification was based on observations of morphological key-characters (i.e., cell shape and sizes, number and positionsof cirri, length of buccal aperture, number and positions ofdorsal kineties, type of argyrome on the dorsal surface) on anappropriate number of living individuals, and on the comparisonof collected data with previous literature (Curds, 1975).

DNA Extraction, MolecularCharacterization of Ciliate BacterialSecondary Endosymbiont andSequencing of Almost Complete 16SrRNA Gene of Rheinheimera sp. EpRS3From a preliminary FISH screening, E. aediculatus strain EASCc1was known to host in its cytoplasm two bacterial symbionts:P. necessarius (Betaproteobacteria), primary endosymbiont,and an unidentified Gammaproteobacterium, secondaryendosymbiont, so for the molecular characterization of thelatter, about 50 starved E. aediculatus strain EASCc1 cells werewashed several times in sterile distilled water and fixed in 70%ethanol. Total genomic DNA extraction was performed usingthe NucleoSpinTM Plant II DNA extraction kit (Macherey-Nagel,Germany), following the protocol for mycelium suggested bythe company. The 16S rRNA gene of the bacterial symbiont wasamplified in a C1000TM Thermal Cycler (BioRad, Hercules,CA, United States) with the TaKaRa ExTaq (TaKaRa Bio Inc.,Otsu, Japan). The16S rRNA gene of the Gammaproteobacteriaendosymbiont was amplified using the primers alfa F19a (5′-CCTGGCTCAGAACGAACG-3′ Vannini et al., 2004) and R1492(5′-GGNWACCTTGTTACGACTT-3′, modified from Lane,1991). Each amplification consisted of 35 cycles of: a preliminarydenaturation step at 94◦C for 3 min, denaturation at 94◦C for30 s, annealing at 50◦C for 30 s and elongation at 72◦C for2 min, and a final elongation step at 72◦C for 6 min. In each PCR

Frontiers in Microbiology | www.frontiersin.org 3 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 4

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

experiment, a control without DNA was always included. Afterevaluation by electrophoresis on 1% agarose gel, PCR productswere purified with EuroGold Cycle-Pure kit (EuroClone1,Milan, Italy) and directly sequenced using prokaryotic universalprimers, 16S R515ND (5′-ACCGCGGCTGCTGGCAC-3′), 16SF785ND (5′-GGATTAGATACCCTGGTA-3′), and 16S F343(5′-TACGGGAGGCAGCAG-3′) (Vannini et al., 2004).

Genomic DNA of Rheinheimera sp. strain EpRS3 wasobtained through thermal lysis. Briefly, an isolated colony(about 108 bacterial cells after 12 h growth, Lodish et al., 2000)was picked up from an overnight culture in solid medium (inthis case, TSA) and dissolved in 50 µl of saline solution (0.9%NaCl); bacterial cells were then submitted to thermal lysis in athermoblock at 95◦C for 10 min, followed by cooling on ice for5 min. The supernatant obtained after 3-min centrifugation atmaximum speed was then placed in a sterile tube and used astemplate DNA for amplification. The amplification of the 16SrRNA gene was performed using the same amplification protocoldescribed for the ciliate symbiont, with universal prokaryoticprimers F7 (5′-AGRGTTYGATYMTGGCTCAG-3′ Vanniniet al., 2004) and R1492 (5′-GGNWACCTTGTTACGACTT-3′,modified from Lane, 1991). After evaluation by electrophoresison 1% agarose gel, PCR products were purified withEuroGold Cycle-Pure kit (EuroClone, Milan, Italy) anddirectly sequenced using prokaryotic universal primers, 16SR515ND (5′-ACCGCGGCTGCTGGCAC-3′), 16S F785ND (5′-GGATTAGATACCCTGGTA-3′), and 16S F343 (5′-TACGGGAGGCAGCAG-3′) (Vannini et al., 2004). The full-lengthsequence of the 16S rRNA gene of Rheinheimera sp. strainEpRS3 was necessary to design the specific FISH probe asdescribed below.

Probe Design for Detection ofRheinheimera sp. EpRS3 and FISHAnalysisA specific probe was designed in silico to detect Rheinheimerasp. EpRS3 using the almost complete characterized 16S rRNAgene sequence (Accession Number MH540131, this work). Thespecificity of probe Rhein443 (5′-TACCAACCCTTCCTCCTC-3′, Tm = 56◦C) was tested in silico both on Ribosomal DatabaseProject (Cole et al., 2013) and TestProbe tool 3.0 (SILVA rRNAdatabase project, Quast et al., 2013). The probe was synthesizedand labeled with Cy3 by Eurofins GMBH (Ebersberg, Germany),and the probe sequence was deposited in ProbeBase (Greuteret al., 2015). FISH experiments were carried out according tothe protocol by Manz et al. (1992) and different formamideconcentrations (0, 10, 20, and 30% v/v) were tested to find theoptimal stringency level for the newly designed probe.

Experimental Set-Up: Treatments of theCulturesThe experimental procedure has been resumed in Figure 1.Rheinheimera sp. EpRS3 was grown overnight on tryptic soybroth medium (TSB) at 30◦C in agitation. After growing,the liquid culture was immediately processed; five different

FIGURE 1 | Schematic representation of the procedure followed for theexperimental design.

treatments were prepared to treat five separate 1-ml ciliate sub-cultures of E. aediculatus strain EASCc1 consisting of 20–30cells each (Supplementary Table S1). Previously, the ciliateswere maintained in starving conditions for 4 days to let themcomplete digestion of their regular bacterial food (Raoultellaplanticola) and to minimize the presence of phagosomeswith Gammaproteobacteria inside. All the sub-cultures receivedtheir specific treatment at the same time. These were theprepared treatments: (A) plain TSB medium (positive control);(B) absence of any treatment (negative control); (C) cell-freefiltered supernatant from EpRS3 bacterial culture (“Supernatant”treatment); (D) liquid culture of EpRS3 bacterial cells with theirgrowth TSB medium (“Tq” treatment); (E) EpRS3 cells withoutTSB growing medium, and resuspended in saline solution (0.9%NaCl in dH2O) (“Pellet” treatment).

Frontiers in Microbiology | www.frontiersin.org 4 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 5

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

Once each ciliate sub-culture was provided with its treatment,eukaryotic cells were counted at defined time points underthe stereomicroscope (×10). The first count was performedimmediately after the beginning of each treatment. Then cellcounts were performed every 60 min, until 24 h. WhenE. aediculatus cells had showed evident signs of suffering, such asa clear swim deceleration, a prolonged absence of cell movementwith ciliature still beating and/or cell number decrease, they wereconsidered close to death and were collected and fixed for TEMand FISH analyses.

In order to study the possible cell damage that ciliateswere facing, cell were fixed immediately before their death.A comparison with negative and positive control specimenswas performed to highlight Euplotes cell structures affected bydifferent EpRS3 treatments. Any potentially observed treatmenteffect on the ciliate obligate endosymbiont P. necessarius andon the secondary endosymbiont “Ca. Nebulobacter yamunensis”was recorded as well.

A total of ten replicate experiments were conducted indifferent days in order to confirm the harmful effect ofRheinheimera sp. EpRS3 vs. the ciliate E. aediculatus EASCc1.Once the effect was confirmed, an experiment was conductedwith the aim to obtain fixed cells (treated and control ciliates)for FISH and TEM analyses: three different culture replicates,monitored up to 24 h, were performed to test the effect of thefive different treatments reported in Figure 1 on the growth andsurvival of the E. aediculatus EASCc1. Variation in ciliate cellnumber was calculated in each case and expressed as % of T0cell number (100%).

Fluorescence in situ Hybridization (FISH)AnalysisFluorescence in situ hybridization analysis was used both toassess the presence of the two different endosymbionts inE. aediculatus confirming preliminary observations using specificprobes already available, and to verify the potential presenceof Rheineimera sp. EpRS3 inside E. aediculatus EASCc1 cells

which underwent culture experiments using the specific probeRhein_443 (this work).

About 15 ciliate cells were randomly collected from bothexperimental and control cultures and were washed three timesin sterile San Benedetto water and separately fixed on slides forFISH experiments as described by Szokoli et al. (2016).

Several different specific probes were used for FISH analysis(Table 1): Poly_862 (Vannini et al., 2005, labeled either withfluorescein or Cy3, green signal and red signal, respectively) todetect P. necessarius, NebProbe_203 (Boscaro et al., 2012, labeledwith Cy3, red signal) to observe “Ca. Nebulobacter yamunensis,”and the newly designed specific probe Rhein443 (labeled withCy3, red signal) to screen for Rheinheimera sp. EpRS3 presence.Two universal probes were employed as control to excludethe presence of other intracellular bacteria, namely Gamma_42(Manz et al., 1992, labeled with Cy3, red signal) to detectGammaproteobacteria, and Eub_338 (labeled with fluorescein orCy3, green signal and red signal, respectively) targeting almosttotal Eubacterial diversity (Amann et al., 1990; Table 1).Thepresence of DAPI in the SlowFade R© Gold Antifade Reagent(Invitrogen) was used as additional control.

The pellet of Rheinheimera sp. EpRS3 from 2 ml, overnightliquid TSB culture (about 1010 cells/ml) was fixed in duplicate forFISH experiments using 2% PFA. 50 µl of the fixed bacterial pelletwere dropped on the slide and hybridized according to Szokoliet al. (2016). FISH was performed using probe Rhein443 (labeledwith Cy3, red signal) and Eub_338 (labeled with fluorescein,green signal) (Table 1) in order to verify the efficiency of thenewly designed probe Rhein443 on Rheinheimera sp. EpRS3.

Images were obtained with a Leica DMR microscope,equipped with an HBO 50W/AC-L2 fluorescent lamp,a Leica DFC490 video camera, and a dedicated SoftwareLeica IM1000 (v.1.0).

Transmission Electron Microscopy (TEM)Transmission electron microscopy analysis was used both toassess the morphology of the two different endosymbionts in

TABLE 1 | List of probes used for FISH experiments reporting probe sequence, their fluorochrome, specificity, formamide concentration, and application.

Name Sequence 5′ to 3′ Fluorochrome Specificity % Formamide inFISH experiment

Reference Treatment

Eub_338 5′-GCTGCCTCCCGTAGGAGT-3′ Fluorescein Almost 90% ofknownEubacteria

0/15 Amann et al., 1990 C+ C−Supernatant TqPellet

NebProbe_203 5′-ATAGCGACTGCCCTAAAG-3′ Cy3 “Ca.Nebulobacteryamunensis”

15 Boscaro et al., 2012 C− C+

Poly_862 5′-GGCTGACTTCACGCGTTA-3′ Fluorescein cy3 Polynucleobactergenus

0 Vannini et al., 2005 C+ C−

Rhein443 5′-TACCAACCCTTCCTCCTC-3′ Cy3 Rheinheimeragenus

0 Present work C− C+Supernatant TqPellet

Gamma_42 5′-GCCTTCCCACATCGTTT-3′ Cy3 Gamma-proteobacteriasubclass

0 Manz et al., 1992 C− C+

C+, TSB medium; C−, no treatment; Supernatant, filtered supernatant of Rheinheimera sp. EpRS3; Tq, EpRS3 + TSB; Pellet, EpRS3 in NaCl 0.9%.

Frontiers in Microbiology | www.frontiersin.org 5 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 6

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

E. aediculatus, and to verify the potential presence of Rheineimerasp. EpRS3 inside E. aediculatus EASCc1 cells which underwentculture experiments. For TEM analysis, eukaryotic cells sampledfrom each of the protist sub-cultures of culture experiments werefixed in a 1:1 mixture of 2% OsO4 in sterile distilled water and2.5% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.4); thencells were ethanol-dehydrated, transferred to 100% acetone, andembedded in an Epon araldite mixture (Modeo et al., 2013a).

After overnight growth in liquid TSB medium the pellet ofRheinheimera sp. EpRS3 (about 1010 bacterial cells/ml) was fixedfor TEM analysis according to Nitla et al. (2018). Prokaryoticcells were fixed in 1.5-ml Eppendorf tubes and all the solutions(fixatives, ethanol, acetone, and Epon araldite embeddingmixture) were directly added. At each step, the Eppendorf tubewith prokaryotic cells was vortexed and centrifuged for 5 minat 5,500 × g; then, after discharging the supernatant, the nextsolution was added, and the pellet was re-suspended.

Ultrathin sections of both eukaryotic and prokaryotic cellTEM preparations were placed on copper grids and stainedwith uranyl acetate and lead citrate prior to observation witha JEOL 100S TEM.

Hydrogen Peroxide Production byRheinheimera sp. EpRS3The production of hydrogen peroxide by Rheinheimera sp.EpRS3 was assessed with a colorimetric assay on agar mediumbased on the Prussian blue-forming reaction (Saito et al.,2007), that was used as described in Chen et al. (2010). Theagar medium allows the identification of hydrogen peroxide-producing bacterial strains by the appearance of dark blue halosaround the colonies, due to the formation of a precipitate.The Prussian blue agar is made as follows (1 L): 1 g ofFeCl3·6H2O dissolved in 50 ml of distilled water (solution A);1 g of potassium hexacyanoferrate(III) K3[FeIII(CN)6] dissolvedin 50 ml of distilled water (solution B); preparation of 900 mlTSA medium; mixing solutions A and B, and pouring the mixtureinto TSA medium; the obtained medium, must be sterilized byautoclaving at 121◦C for 20 min. Bacterial sample were streakedon Prussian blue TSA (PB-TSA), and incubated for 5 days at30◦C, until satisfactory growth; in parallel, different amounts ofhydrogen peroxide were dropped on the same medium, and putunder the same temperature conditions, in order to observe theblue precipitate formation.

RESULTS

Ciliate Identification (SupplementaryFigure S1)Ciliate strain EASCc1 was confirmed in morphologicalinspections as Euplotes aediculatus as perfectly matching thedescription of the species (Curds, 1975); additionally, molecularanalysis based on 18S rRNA gene sequencing confirmedthe species assignation by morphological identification(C. Sigona, pers. commun.).

Bacterial Molecular CharacterizationThe almost complete 16S rRNA gene sequence of theGammaproteobacteria endosymbiont was achieved by directsequencing after PCR on E. aediculatus cells. The sequenceobtained was 1409 bp long (Acc. Number MH608339, this work)and was 100% identical with “Ca. Nebulobacter yamunensis”(GenBank HE794998) already present in NCBI database.

The Rheinheimera sp. EpRS3 strain 16S rRNA gene sequencewas obtained by direct sequencing from the bacterial strainDNA. The sequence was 1455 bp long (Acc. Number MH540131,this work) and 98.27% identical with Rheinheimera pacifica(GenBank NR114230).

Culture Experiments: Cell Counts(Figures 1, 2, and SupplementaryFigure S2)The experimental procedure has been resumed in Figure 1. Inall the replicate experiments, C+ and C− treatments (i.e., thetreatment with plain bacteria TSB medium and the absence ofany treatment, respectively) did not affect the overall healthstatus of ciliates (Figure 2 and Supplementary Figure S2).However, several of the other treatments did affect ciliatesurvival (Figure 2). Supernatant treatment showed conflictingresults depending on the different replicate: in two out often replicates, cell number dramatically decreased between 6and 24 h from T0 (beginning of the treatment); moreover,cells stopped moving 2–4 h from T0. This behavior, withthe same timing, was also recorded in Tq-treated ciliates. Ina third replicate cell number remained constant over time,suggesting that there was not any effect on cell survival. Tqtreatment impacted ciliates in all the ten replicates. After2–4 h from T0, all ciliates stopped movement and showed cellsuffering; in a single replicate ciliate number started a dramaticdecreasing already after 1 h from T0. In all the replicates, thealmost complete disappearance of treated ciliate sub-culture wasrecorded between 6 and 24 h.

Some Tq-treated cells were isolated soon after stoppingmovement and transferred to pure freshwater in order toroughly test whether the treatment effect was immediate and/orpermanent. They apparently recovered well after some minutes,but it is not possible to assert if they were able to survivefor a longer time and also to divide in the light of the TEMobservation which reported on heavy Euplotes/endosymbiont celldamages (see later).

Pellet treatment did not affect protozoan survival in any ofthe ten replicate experiments, and the ciliate sub-culture survivedover time: cells showed a healthy aspect, and, in some cases, theyduplicated with an increment of number.

Rhein_443 Probe DesignIn the present study the probe Rhein_443 (5′-TACCAACCCTTCCTCCTC-3′, Tm = 56◦C) was tested forits optimum formamide concentration by applying formamidefrom 0 to 30%, and the highest signal intensity was recordedat 0%. Its specificity was verified in silico both on RibosomalDatabase Project (Cole et al., 2013) and on TestProbe tool

Frontiers in Microbiology | www.frontiersin.org 6 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 7

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

FIGURE 2 | Culture experiment. Ten different replicates (indicated by ten different colors), three of them monitored over 24 h, performed to test the effect ofRheinheimera sp. EpRS3 on the growth, and survival of E. aediculatus EASCc1. Supernatant, Tq, Pellet, and C– treatments according to text. Variation in cellnumber is reported as % with respect to T0 cell number (100%).

3.0 (SILVA rRNA database project Quast et al., 2013). Thenewly designed probe recognized in silico four non-targetsequences, but its specificity was always verified during FISHexperiments using the universal probe Gamma_42 targetingGammaproteobacteria (Table 1).

FISH Experiments (Figure 3 andSupplementary Figures S1, S3, S4)In the cytoplasm of the ciliate, the endosymbiont P. necessariuswas well visible in culture experiments under the light microscopedue to its large size and high abundance (Supplementary FiguresS1A,B). Preliminary FISH experiments on the ciliates withPoly_862, Gamma_42, and NebProb_203 probes highlighted thepresence in the cytoplasm of two distinct endosymbiotic bacteria:the betaproteobacterium P. necessarius (Supplementary FiguresS1C,D) and the gammaproteobacterium “Ca. Nebulobacteryamunensis” (Supplementary Figures S1E,F). FISH experimentson Rheinheimera sp. EpRS3 bacterial cells produced positivesignals with both EUB_338_Fluo (Supplementary Figure S3A)and Rhein443_cy3 (Supplementary Figure S3B) probes. FISHexperiments on C− and C+ ciliate subcultures hybridized withPoly_862, Gamma_42, and NebProb_203 probes, confirmedthe presence of the two bacterial endosymbionts in ciliatecytoplasm. On the contrary, FISH experiments on the same

two ciliate subcultures with Eub_338 and Rhein443 gavepositive signals only with the first probe, revealing theabsence of Rheinheimera sp. EpRS3 cells (C−, Figures 3A,B;C+, data not shown).

In Supernatant-treated sub-culture ciliates Rhein443 probegave negative signal indicating the absence of Rheinheimera sp.EpRS3 (Figures 3C,D). In Pellet-treated ciliates, Rhein443 probehighlighted the presence of Rheinheimera sp. EpRS3 inside largefood vacuoles (Figures 3E,F).

On ciliates of the Tq-treated subculture, Rhein443 proberevealed positive signals in the cytoplasm, indicating thepresence of Rheinheimera sp. EpRS3 (Figures 3G,H).It is worth noting that these positive signals indicatedthe presence of Rheinheimera sp. EpRS3 both in smallphagosomes and free in the cytoplasm, i.e., not included inphagosomes. Additionally, some Rheinheimera sp. EpRS3bacteria were targeted outside the ciliate (SupplementaryFigure S4), divergently from what observed in all otherFISH experiments.

TEM Observation (Figures 4–8)A preliminary TEM experiment was carried out on Rheinheimerasp. EpRS3 in order to obtain the first morphological-ultrastructural data on these bacterial cells (Figure 4). Cells

Frontiers in Microbiology | www.frontiersin.org 7 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 8

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

generally appeared rod-shaped and encircled by a doublemembrane. They showed a linear outline and a highly electron-dense peripheral region; cell electron-density decreased in theinner structure, leading to a clear central core. Cells were fixedduring their stationary growth phase and did not show thepresence of different bacterial morphologies, which might occurwith diverse life cycle stage. No bacterial flagellum was observed;however, the lack of flagella could also be due to TEM procedure.Bacterial cell dimensions ranged from ∼ 1.5 µm × 0.8 µm up to∼ 4 µm× 0.8 µm.

TEM observation on E. aediculatus cells confirmed theresults of FISH experiments. As expected, in C− cells (notreatment) the presence of two endosymbionts was observed,i.e., P. necessarius (maximum size: ∼ 6.5 µm × 0.4 µm; veryabundant) (Figure 5A) and “Ca. Nebulobacter yamunensis”(size: ∼ 1.5 µm × 0.6 µm; rare) (Figures 5B,C). Both theendosymbionts showed a double membrane as typical ofGram-negative bacteria. P. necessarius cells were characterizedby a slender aspect, an undulating external membrane, anon-homogeneous, generally electron dense cytoplasm,

FIGURE 3 | FISH experiment on E. aediculatus EASCc1 cells (0% formamide) using Eub_338 in green (detected almost 90% of known Eubacteria) and Rhein443 inred (detected Rheinheimera genus). (A,B) Negative control of culture experiment (i.e., not-treated ciliates). (C,D) Supernatant treatment. (E,F) Pellet treatment.Arrow, phagosome. (G,H) Tq treatment: signals of Rhein443_cy3 probe corresponding to Rheinheimera sp. EpRS3 cells (arrow). Scale bars: 10 µm.

Frontiers in Microbiology | www.frontiersin.org 8 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 9

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

with some inner clearer regions where the presence ofnucleoids sometimes is visible. “Ca. Nebulobacter yamunensis”usually appeared squatter and showed a homogeneous, clearinner cytoplasm, which becomes more electron dense inits periphery. A clear halo was sometimes visible aroundP. necessarius cells.

Positive control cells (C+, treatment with sterile TSBmedium) showed no ultrastructural differences with C−cells (data not shown), as bacteria growth medium itselfdid not affect ciliate cells in any possible way. By TEMobservation, E. aediculatus cells treated with Supernatanttreatment (Rheinheimera sp. EpRS3 cell-free supernatant),showed a slightly vacuolized cytoplasm (Figures 6A,B),while mitochondria and plasma membrane appeared notaffected (Figure 6C). Ciliates still hosted in the cytoplasmmany P. necessarius bacterial endosymbionts, but several ofthem showed clear suffering signs, such as high cytoplasmvacuolation and degradation, being altered at different grades(Figures 6A,C,D). Representatives of “Ca. Nebulobacteryamunensis” generally showed a rather regular low abundanceand appearance (Figure 6B).

Several very electrodense bodies enclosed in vacuoles wereobserved in the cytoplasm of both Supernatant (Figure 6D)and Tq (see later) treated cells; these are obliquely- or cross-sectioned, highly degraded P. necessarius cells, as their abundanceand general appearance fit with those of longitudinally sectioned,degraded P. necessarius endosymbionts observed in thesame TEM sections.

In Tq-treated ciliates, plasma membrane was still wellconserved, while majority of P. necessarius endosymbiontswere vacuolized and degraded as much as in Supernatant-treated cells. (Figure 7A). Autophagosomes were also presentin the cytoplasm, in some cases including P. necessarius cells(Figure 7B). The endosymbiont “Ca. Nebulobacter yamunensis”was apparently not affected by the treatment (Figure 7C). Unlikewhat observed in Supernatant-treated cell, in Tq-treated ciliatesmitochondria were very degraded as well; many of them werebroken and, in some cases, they appeared to have undergone akind of membrane disassembly (Figures 7A,B,D).

In Tq treated-cells, EpRS3 bacterial cells were observedboth enclosed (Figure 7F) and not enclosed in phagosomes(i.e., free in the cytoplasm) (Figures 7G–I). They couldbe identified based on shape, dimensions, and appearancethat distinguished them both from P. necessarius and “Ca.Nebulobacter yamunensis” endosymbionts. These resultsconfirmed FISH experiment findings.

Ciliate cells treated with Pellet treatment showedcytoplasm and endosymbiont appearance similar to C− cells(Figures 8A,B). In Pellet-treated ciliates many Rheinheimera sp.EpRS3 cells (R) were observed inside phagosomes, in differentdigestion stages (Figure 8B).

Hydrogen Peroxide Production by EpRS3(Supplementary Figure S5)Prussian blue agar medium assay revealed that RheinheimeraEpRS3 was able to grow on the modified PB-TSA medium.The growth after 5 days incubation did not reveal anyhydrogen peroxide production by Rheinheimera EpRS3; this wasevidenced by the absence of the blue halo around bacterialcells (Supplementary Figure S5, right side). This was alsoconfirmed through comparison with the blue halos appearedaround hydrogen peroxide dropped at different concentration onthe same modified medium (Supplementary Figure S5, left side).

DISCUSSION

Antagonistic interactions in complex microbial communitieshave been largely investigated in the past years as meansof competition for space and resources (e.g., Hibbing et al.,2010; Becker et al., 2012). Antagonistic interactions amongbacterial species are often expressed by means of the releaseof toxic/antagonistic compounds, that are used to prevent theentrance of other bacterial microorganisms, by creating aninhospitable zone for competitors (Stubbendieck and Straight,2016; Stubbendieck et al., 2016). Bacteria are largely studiedfor their ability to produce metabolites with a biocidal effectagainst other bacteria (Maida et al., 2016; Chiellini et al., 2017)

FIGURE 4 | TEM observation on the pellet of Rheinheimera sp. EpRS3 processed after overnight liquid TSB culture (stationary growth phase). (A) Cells showdifferent size and different aspect due to pellet sectioning. (B) Some cells are dividing. Scale bars: 1 µm.

Frontiers in Microbiology | www.frontiersin.org 9 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 10

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

FIGURE 5 | TEM observation on E. aediculatus EASCc1 cytoplasm withouttreatment (C– sub-culture specimens). (A) Some Polynucleobacternecessarius (P) endosymbionts adjacent to macronucleus (Ma). (B) “Ca.Nebulobacter yamunensis” (N) and P. necessarius. (C) Another specimen of“Ca. Nebulobacter yamunensis.” Scale bars: 1 µm.

and against fungi (Magnusson et al., 2003). Antagonisticinteractions between bacteria and protists, are instead a stillpoorly investigated, but ecologically relevant field (Matzet al., 2004a). The ecological role of this ability might find

its explanation as an attempt to avoid protist grazing, andhelp improving the survival of bacterial population in manyenvironments (e.g., Jousset et al., 2006).The strategies to surviveagainst protist predation include the production of bioactivemetabolites (Matz et al., 2004a), bacterial motility characteristics(Matz and Jürgens, 2005), the biological properties of thecell external surface (Wildschutte et al., 2004), and thecommunication among cells (Matz et al., 2004a).

In the present research, the antagonistic interaction betweenthe freshwater ciliated protozoan E. aediculatus strain EASCc1and the antimicrobial producer endophytic Rheinheimera sp.EpRS3 has been documented in culture experiments, andconfirmed for the first time by means of FISH and TEMexperiments. Firstly, the lack of any variation in protists cellnumber observed in the culture experiments (C+ and C−treatments) allows to hypothesize that all the effects highlightedin ciliates treated with the other three treatments (Supernatantand Tq) were due to the presence of Rheinheimera sp. EpRS3bacteria and to one or more putative bioactive molecule(s) theyproduce (Figure 2). Moreover, the positive signals of Gamma,Poly, and NebProb FISH probes in the cytoplasm of E. aediculatuscells provided to C+ and C− treatments (data not shown), alsoconfirmed that the bacterial endosymbionts were not affected byeither the TSB medium or the cell manipulation occurred duringthe experimental procedures.

In FISH experiments, the absence of signal by the probeRhein443 in E. aediculatus cells of C− (Figure 3B), C+ (datanot shown), and Supernatant (Figure 3D) treatments, andthe positive signals observed after Pellet and Tq treatments(Figures 3F,H), indicate the presence of living Rheinheimerasp. EpRS3 cells only in ciliates treated with the latter twotreatments. After Tq treatment, at difference with Pellettreatment where bacteria appear enclosed in phagosomes andin different digestion stages (Figure 3F), only a few bacterialcells are enclosed in small phagosomes, while most of themare free in the cytoplasm of treated ciliates (Figure 3H).These results have been confirmed by TEM observation(Figures 7G–I) and are in accordance with previous studiesdisclosing that some bacteria are capable of surviving to ciliatedigestion in food vacuoles (King et al., 1988). In particular,Gammaproteobacteria and Alphaproteobacteria are reported asprevalent as digestion-resistant bacteria in ciliated protozoa(Gong et al., 2016). It could be hypothesized that Rheinheimerasp. EpRS3 cells can take advantage of suffering ciliates andeasily escape phagosomes, thus entering the cytoplasm andstarting to produce bioactive molecule(s) with a biocidal effect.Interestingly, also in Tq-treated Euplotes samples observedat TEM, dividing Rheinheimera sp. EpRS3sp. specimens havebeen observed free in ciliate cytoplasm, which indicates thatthese bacteria are healthy and might potentially colonizethe host cytoplasm as an endosymbiont. However, ciliatemitochondrial degradation has been observed at TEM in Tq-treated Euplotes, which could be explained by the effect ofRheinheimera’s bioactive molecule(s). It can be speculated thatthese might be produced during the entrance of Rheinheimeracells in the ciliate cytoplasm, or before the entrance of thebacterium in the suffering ciliates. It cannot be ruled out

Frontiers in Microbiology | www.frontiersin.org 10 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 11

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

the possibility that some autophagy phenomena concerningorganelles such as mitochondria occurred in ciliate cytoplasmas well (Szokoli et al., 2016). Moreover, according to probesignals in FISH experiments, Rheinheimera sp. EpRS3 couldpossibly leave the ciliate when the latter is suffering, assuggested by the positive FISH signals of Rhein443_cy3 probeobserved on ciliate surface (Supplementary Figure S4). Thisinteresting result, if validated by further data, could supportthe hypothesis that healthy bacterial cells inside ciliates mightperhaps reintroduce themselves into the environment, as itis known for other infectious ciliate endosymbionts (e.g.,Holospora) (Potekhin et al., 2018).

Concerning the endosymbionts of E. aediculatus, afterSupernatant and Tq treatments, P. necessarius (highly abundantendosymbiont) appeared very damaged (Figures 6A,C,D,7A,B) while “Ca. Nebulobacter yamunensis” (less abundantendosymbiont) seemed not affected (Figures 6C, 7C) in TEM-processed ciliates. The symbiotic relationship between Euplotesand Polynucleobacter is well-known, as it is an obligatemutualistic endosymbiont (Heckmann, 1983; Heckmann andSchmidt, 1987; Görtz and Schmidt, 2004), while up to nownothing is known about the nature of the symbiotic associationbetween Euplotes and “Ca. Nebulobacter yamunensis” as the

latter has been only molecularly characterized (Boscaro et al.,2012). This is the first time the ultrastructure of this bacteriumhas been investigated and the present study gives hints onthe not strict bound between the ciliate host and “Ca.Nebulobacter yamunensis,” as the endosymbiont is not affectedby ciliate suffering.

In the light of the present findings, the following hypothesiscan be formulated to explain Rheinheimera sp. EpRS3 behaviorin the presence of the protist ciliate E. aediculatus. RheinheimeraEpRS3 sp. produces one or more different molecule(s) withan evident deleterious effect on E. aediculatus; these moleculesare only present in the Tq treatment (bacteria plus culturemedium). The Pellet treatment does not contain any trace of thesebioactive molecules with deleterious potential; indeed, EpRS3cells are ingested and digested by the ciliate together with foodbacteria (R. planticola) without any apparent problem. In thiscase, Rheinheimera is treated as a food source by the protist,which can survive. On the other side, the filtered Supernatant,free of bacterial cells, is not sufficient to strongly affect the wholeE. aediculatus population survival. This is demonstrated (i) bythe cell counts on E. aediculatus repeated culture experiments,showing that only in a few cases the cell number decreasesand (ii) by the TEM observation showing vacuolization and

FIGURE 6 | TEM observation on E. aediculatus EASCc1 treated with Supernatant treatment. (A) Ciliate cytoplasm vacuolized and a degraded P. necessarius (P).(B) Plasma membrane and mitochondria appear regular. (C) After Supernatant treatment “Ca. Nebulobacter yamunensis” (N) is not affected, while P. necessarius isaffected (P). (D) Some P. necessarius endosymbionts at different stages of degradation. Scale bars: 1 µm.

Frontiers in Microbiology | www.frontiersin.org 11 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 12

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

FIGURE 7 | TEM investigation on E. aediculatus EASCc1 after Tq treatment. (A) Degraded mitochondria (m) and a vacuolized, highly degraded P. necessarius (P) inlongitudinal section. (B) P. necessarius cells included in autophagosomes (a). (C) “Ca. Nebulobacter yamunensis” (N) and some damaged mitochondria. (D,E)Broken, highly degraded mitochondria. (F) Rheinheimera sp. EpRS3 cells (R) inside a phagosome. (G) Rheinheimera sp. strain EpRS3 free in ciliate cytoplasm. (H) Adividing specimen of Rheinheimera sp. (R) and a degraded mitochondrion (m). (I) Rheinheimera sp. free in cytoplasm in longitudinal section (R). Scale bars: 1 µm.

FIGURE 8 | (A) TEM investigation on E. aediculatus EASCc1 after Pellet treatment. Cell aspect resembles that of C– cells. Ma, macronucleus; P, P. necessariusendosymbiont. (B) A large phagosome (ph) with bacteria undergoing digestion. Scale bars: 1 µm.

Frontiers in Microbiology | www.frontiersin.org 12 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 13

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

degradation of some of the P. necessarius endosymbionts, but nodamage of fundamental ciliate organelles such as mitochondria.Finally, Tq treatment helped to highlight a possible explanationabout the harmful effect exerted by EpRS3 on E. aediculatus.Rheinheimera sp. cells are initially ingested and stored in thephagosomes, treated as a food source by the protozoa. But theTq treatment, might contain also all the bioactive moleculesthat are produced from the bacterium after overnight growth,which might damage host mitochondria leading Euplotes andits mutualistic symbiont to death. It is known that symbioticP. necessarius has a very reduced genome and depends on its hostfor many metabolic pathways (Boscaro et al., 2013a). This couldexplain why P. necessarius suffers in the Tq treatment in presenceof degraded mitochondria, while in the Supernatant treatmentthe bacterial endosymbionts decline probably due to the presenceof bioactive molecules, not affecting host mitochondria.

The proposed scenario might be part of all those strategiesused by bacteria in order to survive against protist predation,asserted through grazing pressure. Although further experimentsshould be performed to assert it with certainty, our data suggestedthat the toxicity of Rheinheimera EpRS3 might not be explainedby means of production of hydrogen peroxides, contrary towhat has already been reported in other Rheinheimera strainsshowing similar effects on bacteria and eukaryotes (Chen et al.,2010). The described hypothesis might be included among thosestrategies previously described as “post-ingestion adaptations”(Matz and Kjelleberg, 2005). In this case, the internalizedbacterial cells are enclosed in digestive vacuoles, and face with anextremely acidophilic environment, as well as with the presenceof degradative enzymes to survive. Sometimes, the resistance ofbacteria in such hostile environment can be attributed to peculiarbacterial protective features such as in the case of the microbialS-layers of Synechococcus cells in the ciliate Tetrahymena (Koval,1993). In this case, further investigation will be required tofind out molecular mechanisms related to the resistance of thebacterium inside digestive vacuoles.

The present study is only preliminary, and the speculativehypotheses here proposed definitely need further investigation tobe confirmed. We are planning to address the attention on thepossible characterization of the bioactive molecule(s) producedby the bacterium by means of an integrated multidisciplinarystudy approach. On the light of the available literature, aninteresting and still unexplored field is represented by theputative role of the Rheinheimera sp. EpRS3 TolC in themechanisms leading to the interaction with the protozoan cell,

as previously documented in Legionella pneumophila towardParamecium tetraurelia (Nishida et al., 2018). Indeed, it hasbeen previously demonstrated that EpRS3 genome harborsseveral genes encoding for components of efflux pump systems,like AcrB, belonging to the AcrAB/TolC system, or MdtBand MdtC, these last forming a heteromultimer complex, asubunit of MdtABC-TolC efflux pump (Presta et al., 2017). Thisinformation will help in elucidating the molecular mechanismsleading to the eukaryotic cell structures damage, to protist death,and to the possible re-entry of Rheinheimera sp. EpRS3 intothe environment.

AUTHOR CONTRIBUTIONS

CC, CP, LM, GP, and RF designed the research. CC and CPperformed the culture experiments. CP and LM performed theTEM experiments. CP and OL performed the FISH experiments.CC, CP, OL, and LM interpreted the results and wrote themanuscript. GP, RF, CB, and CF revised the manuscript. GP,CB, and RF financed the research. All authors critically read andapproved the manuscript.

FUNDING

This research was partially supported by Progetti di Ricerca diAteneo, PRA_2016_58, University of Pisa to GP.

ACKNOWLEDGMENTS

The authors gratefully acknowledge: Cristiana Sigona forproviding E. aediculatus strain EASCc1, Fabrizio Erra andSimone Pacini for their help with fluorescence microscopeobservation, Venkata Mahesh Nitla for help with microscopeobservation of cultures, Simone Gabrielli for pictures and figureelaboration; and Franco Verni for discussion on TEM results.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00510/full#supplementary-material

REFERENCESAbd, H., Wretlind, B., Saeed, A., Idsund, E., Hultenby, K., and

Sandström, G. (2008). Pseudomonas aeruginosa utilises its type IIIsecretion system to kill the free-living amoeba Acanthamoeba castellanii.J. Eukaryot. Microbiol. 55, 235–243. doi: 10.1111/j.1550-7408.2008.00311.x

Amann, R. I., Binder, B. J., Olson, R. J., Chisholm, S. W., Devereux, R., andStahl, D. A. (1990). Combination of 16S rRNA-targeted oligonucleotide probeswith flow cytometry for analysing mixed microbial populations. Appl. Environ.Microbiol. 56, 1919–1925.

Becker, J., Eisenhauer, N., Scheu, S., and Jousset, A. (2012). Increasing antagonisticinteractions cause bacterial communities to collapse at high diversity. Ecol. Lett.15, 468–474. doi: 10.1111/j.1461-0248.2012.01759.x

Boscaro, V., Felletti, M., Vannini, C., Ackerman, M. S., Chain, P. S., Malfatti, S.,et al. (2013a). Polynucleobacter necessarius, a model for genome reduction inboth free-living and symbiotic bacteria. PNAS 110, 18590–18595. doi: 10.1073/pnas.1316687110

Boscaro, V., Fokin, S. I., Schrallhammer, M., Schweikert, M., and Petroni, G.(2013b). Revised systematics of Holospora-like bacteria and characterization of“Candidatus Gortzia infectiva,” a novel macronuclear symbiont of Parameciumjenningsi. Microb. Ecol. 65, 255–267. doi: 10.1007/s00248-012-0110-2

Frontiers in Microbiology | www.frontiersin.org 13 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 14

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

Boscaro, V., Petroni, G., Ristori, A., Verni, F., and Vannini, C. (2013c). ‘CandidatusDefluviella procrastinata’ and ‘Candidatus Cyrtobacter zanobii’, two novel ciliateendosymbionts belonging to the ‘Midichloria clade’. Microb. Ecol. 65, 302–310.doi: 10.1007/s00248-012-0170-3

Boscaro, V., Vannini, C., Fokin, S. I., Verni, F., and Petroni, G. (2012).Characterization of “Candidatus Nebulobacter yamunensis” from thecytoplasm of Euplotes aediculatus (Ciliophora, Spirotrichea) and emendeddescription of the family Francisellaceae. Syst. Appl. Microbiol. 35, 432–440.doi: 10.1016/j.syapm.2012.07.003

Castelli, M., Sabaneyeva, E., Lanzoni, O., Lebedeva, N., Floriano, A. M., Gaiarsa, S.,et al. (2018a). The extracellular association of the bacterium “CandidatusDeianiraea vastatrix” with the ciliate Paramecium suggests an alternativescenario for the evolution of Rickettsiales∗. bioRxiv [Preprint]. doi: 10.1101/479196

Castelli, M., Serra, V., Senra, M. V. X., Basuri, C. K., Soares, C. A. G., Fokin,S. I., et al. (2018b). The hidden world of Rickettsiales symbionts: "CandidatusSpectririckettsia obscura," a novel bacterium found in Brazilian and IndianParamecium caudatum. Microb. Ecol. doi: 10.1007/s00248-018-1243-8 [Epubahead of print].

Chen, W. M., Lin, C. Y., and Sheu, S. Y. (2010). Investigating antimicrobialactivity in Rheinheimera sp. due to hydrogen peroxide generatedby l-lysine oxidase activity. Enzyme Microb. Tech. 46, 487–493.doi: 10.1016/j.enzmictec.2010.01.006

Chiellini, C., Maida, I., Emiliani, G., Mengoni, A., Mocali, S., Fabiani, A., et al.(2014). Endophytic and rhizospheric bacterial communities isolated from themedicinal plants Echinacea purpurea and Echinacea angustifolia. Int. Microbiol.17, 165–174. doi: 10.2436/20.1501.01.219

Chiellini, C., Maida, I., Maggini, V., Bosi, E., Mocali, S., Emiliani, G., et al.(2017). Preliminary data on antibacterial activity of Echinacea purpurea-associated bacterial communities against Burkholderia cepacia complex strains,opportunistic pathogens of cystic fibrosis patients. Microbiol. Res. 196, 34–43.doi: 10.1016/j.micres.2016.12.001

Cho, K. M., Hong, S. Y., Lee, S. M., Kim, Y. H., Kahng, G. G., Lim, Y. P., et al. (2007).Endophytic bacterial communities in ginseng and their antifungal activityagainst pathogens. Microb. Ecol. 54, 341–351. doi: 10.1007/s00248-007-9208-3

Cole, J. R., Wang, Q., Fish, J. A., Chai, B., McGarrell, D. M., Sun, Y., et al. (2013).Ribosomal database project: data and tools for high throughput rRNA analysis.Nucleic Acids Res. 42, D633–D642. doi: 10.1093/nar/gkt1244

Curds, C. R. (1975). A guide to the species of the genus Euplotes (Hypotrichida,Ciliatea). Bull. Br. Mus. Zool. 28, 1–61.

Fokin, S. I., Schrallhammer, M., Chiellini, C., Verni, F., and Petroni, G. (2014).Free-living ciliates as potential reservoirs for eukaryotic parasites: occurrenceof a trypanosomatid in the macronucleus of Euplotes encysticus. Parasite Vector7:203. doi: 10.1186/1756-3305-7-203

Fokin, S. I., Serra, V., Ferrantini, F., Modeo, L., and Petroni, G. (2019). “CandidatusHafkinia simulans” gen. nov., sp. nov., a novel Holospora-like bacteriumfrom the macronucleus of the rare brackish water ciliate Frontonia salmastra(Oligohymenophorea, Ciliophora): multidisciplinary characterization of thenew endosymbiont and its host. Microb. Ecol. doi: 10.1007/s00248-018-1311-0 [Epub ahead of print].

Gong, J., Qing, Y., Fu, R., Su, L., Zhang, X., and Zhang, Q. (2016). Protist-bacteriaassociations: Gammaproteobacteria and Alphaproteobacteria are prevalentas digestion-resistant bacteria in ciliated protozoa. Front. Microbiol. 7:498.doi: 10.3389/fmicb.2016.00498

Görtz, H. D., and Schmidt, H. J. (2004). “Caedibacter, Holosporaceae,Lyticum, Paracaedibacter, Pseudocaedibacter, Pseudolyticum, Tectibacterand Polynucleobacter,” in Bergey’s Manual of Systematic Bacteriology, Vol. 2,eds G. M. Garrity, D. L. Brenner, N. R. Krieg, and J. T. Staley (New York, NY:Springer-Verlag).

Greuter, D., Loy, A., Horn, M., and Rattei, T. (2015). ProbeBase–an online resourcefor rRNA-targeted oligonucleotide probes and primers: new features 2016.Nucleic Acids Res. 44, D586–D589. doi: 10.1093/nar/gkv1232

Heckmann, K. (1983). Endosymbionts of Euplotes∗. Int. Rev. Cytol. (Suppl. 14),111–144.

Heckmann, K., and Schmidt, H. J. (1987). Polynucleobacter necessarius gen. nov.,sp. nov., an obligately endosymbiotic bacterium living in the cytoplasm ofEuplotes aediculatus. Int. J. Syst. Bacteriol. 37, 456–457. doi: 10.1099/00207713-37-4-456

Hibbing, M. E., Fuqua, C., Parsek, M. R., and Peterson, S. B. (2010). Bacterialcompetition: surviving and thriving in the microbial jungle. Nat. Rev. Microbiol.8:15. doi: 10.1038/nrmicro2259

Hudson, J. B. (2012). Applications of the phytomedicine Echinacea purpurea(Purple Coneflower) in infectious diseases. J. BioMed. Res. Int. 2012:769896.

Jousset, A., Lara, E., Wall, L. G., and Valverde, C. (2006). Secondary metaboliteshelp biocontrol strain Pseudomonas fluorescens CHA0 to escape protozoangrazing. Appl. Environ. Microbiol. 72, 7083–7090. doi: 10.1128/AEM.00557-06

Kalinovskaya, N. I., Romanenko, L. A., and Kalinovsky, A. I. (2017).Antibacterial low-molecular-weight compounds produced by the marinebacterium Rheinheimera japonica KMM 9513 T. Antonie Van Leeuwenhoek 110,719–726. doi: 10.1007/s10482-017-0839-1

King, C. H., Shotts, E. B., Wooley, R. E., and Porter, K. G. (1988). Survival ofcoliforms and bacterial pathogens within protozoa during chlorination. Appl.Environ. Microbiol. 54, 3023–3033.

Koval, S. F. (1993). “Predation on bacteria possessing S-layers,” in Advances inBacterial Paracrystalline Surface Layers, eds T. J. Beveridge and S. F. Koval(New York, NY: Plenum Press), 85–92. doi: 10.1007/978-1-4757-9032-0_9

Lane, D. J. (1991). “16S/23S rRNA sequencing,” in Nucleic Acid Techniques inBacterial Systematics, eds E. Stackebrandt and M. Goodfellow (New York, NY:Wiley), 115–147.

Lanzoni, O., Sabaneyeva, E., Modeo, L., Castelli, M., Lebedeva, N., Verni, F.,et al. (2019). Diversity and environmental distribution of the cosmopolitanendosymbiont “Candidatus Megaira”. Sci. Rep. 9:1179. doi: 10.1038/s41598-018-37629-w

Ligon, J. M., Hill, D. S., Hammer, P. E., Torkewitz, N. R., Hofmann, D.,Kempf, H. J., et al. (2000). Natural products with antifungal activity fromPseudomonas biocontrol bacteria. Pest Manag. Sci. 56, 688–695. doi: 10.1002/1526-4998(200008)56:8<688::AID-PS186>3.0.CO;2-V

Linder, J. W. K. E. (1999). Free-living amoebae protecting Legionella in water:the tip of an iceberg? Scand. J. Infect. Dis. 31, 383–385. doi: 10.1080/00365549950163833

Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., and Darnell, J.(2000). “Growth of microorganisms in culture,” in Molecular Cell Biology, 4thEdn, ed. H. Lodish (New York, NY: W. H. Freeman).

Lovejoy, C., Bowman, J. P., and Hallegraeff, G. M. (1998). Algicidal effects of a novelmarine Pseudoalteromonas isolate (Class Proteobacteria, Gamma Subdivision)on harmful algal bloom species of the genera Chattonella, Gymnodinium, andHeterosigma. Appl. Environ. Microbiol. 64, 2806–2813.

Magnusson, J., Ström, K., Roos, S., Sjögren, J., and Schnürer, J. (2003). Broadand complex antifungal activity among environmental isolates of lactic acidbacteria. FEMS Microbiol. Lett. 219, 129–135. doi: 10.1016/S0378-1097(02)01207-7

Maida, I., Chiellini, C., Mengoni, A., Bosi, E., Firenzuoli, F., Fondi, M., et al. (2016).Antagonistic interactions between endophytic cultivable bacterial communitiesisolated from the medicinal plant Echinacea purpurea. Environ. Microbiol. 18,2357–2365. doi: 10.1111/1462-2920.12911

Manz, W., Amann, R. I., Ludwig, W., Wagner, M., and Schleifer, K. H.(1992). Phylogenetic oligodeoxynucleotide probes for the major sub- classesof Proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15, 593–600.doi: 10.1016/S0723-2020(11)80121-9

Matz, C., Bergfeld, T., Rice, S. A., and Kjelleberg, S. (2004a). Microcolonies, quorumsensing, and cytotoxicity determine the survival of Pseudomonas aeruginosabiofilms exposed to protozoan grazing. Environ. Microbiol. 6, 218–226.

Matz, C., Deines, P., Boenigk, J., Arndt, H., Eberl, L., Kjelleberg, S., et al. (2004b).Impact of violacein-producing bacteria on survival and feeding of bacterivorousnanoflagellates. Appl. Environ. Microbiol. 70, 1593–1599.

Matz, C., and Jürgens, K. (2005). High motility reduces grazing mortality ofplanktonic bacteria. Appl. Environ. Microbiol. 71, 921–929. doi: 10.1128/AEM.71.2.921-929.2005

Matz, C., and Kjelleberg, S. (2005). Off the hook–how bacteria survive protozoangrazing. Trends Microbiol 13, 302–307. doi: 10.1016/j.tim.2005.05.009

Mengoni, A., Maida, I., Chiellini, C., Emiliani, G., Mocali, S., Fabiani, A., et al.(2014). Antibiotic resistance differentiates Echinacea purpurea endophyticbacterial communities with respect to plant organs. Res. Microbiol. 165, 686–694. doi: 10.1016/j.resmic.2014.09.008

Modeo, L., Fokin, S. I., Boscaro, V., Andreoli, I., Ferrantini, F., Rosati, G., et al.(2013a). Morphology, ultrastructure, and molecular phylogeny of the ciliate

Frontiers in Microbiology | www.frontiersin.org 14 March 2019 | Volume 10 | Article 510

fmicb-10-00510 March 22, 2019 Time: 20:29 # 15

Chiellini et al. Rheinheimera sp. EpRS3 Killing Euplotes aediculatus

Sonderia vorax with insights into the systematics of order Plagiopylida. BMCMicrobiol. 13:40. doi: 10.1186/1471-2180-13-40

Modeo, L., Petroni, G., Lobban, C. S., Verni, F., and Vannini, C. (2013b).Morphological, ultrastructural, and molecular characterization of Euplotidiumrosati n. sp. (Ciliophora, Euplotida) from Guam. J. Eukaryot. Microbiol. 60,25–36. doi: 10.1111/jeu.12003

Nishida, T., Hara, N., Watanabe, K., Shimizu, T., Fujishima, M., and Watarai, M.(2018). Crucial role of Legionella pneumophila TolC in the inhibition of cellulartrafficking in the protistan host Paramecium tetraurelia. Front. Microbiol. 9:800.doi: 10.3389/fmicb.2018.00800

Nitla, V., Serra, V., Fokin, S. I., Modeo, L., Verni, F., Sandeep, B. V., et al.(2018). Critical revision of the family Plagiopylidae (Ciliophora: Plagiopylea),including the description of two novel species, Plagiopyla ramani and Plagiopylanarasimhamurtii, and redescription of Plagiopyla nasuta Stein, 1860 fromIndia∗. Zool. J. Linn. Soc. zly041. doi: 10.1093/zoolinnean/zly041/5095288

Pandey, A., Palni, L. M. S., and Coulomb, N. (1997). Antifungal activity of bacteriaisolated from the rhizosphere of established tea bushes. Microbiol. Res. 152,105–112. doi: 10.1016/S0944-5013(97)80030-4

Potekhin, A., Schweikert, M., Nekrasova, I., Vitali, V., Schwarzer, S., Anikina, A.,et al. (2018). Complex life cycle, broad host range and adaptation strategy ofthe intranuclear Paramecium symbiont Preeria caryophila comb. nov. FEMSMicrobiol. Ecol. 94:fiy076. doi: 10.1093/femsec/fiy076

Presta, L., Bosi, E., Fondi, M., Maida, I., Perrin, E., Miceli, E., et al. (2017).Phenotypic and genomic characterization of the antimicrobial producerRheinheimera sp. EpRS3 isolated from the medicinal plant Echinacea purpurea:insights into its biotechnological relevance. Res. Microbiol. 168, 293–305. doi:10.1016/j.resmic.2016.11.001

Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., et al. (2013). TheSILVA ribosomal RNA gene database project: improved data processing andweb-based tools. Nucleic Acids Res. 41, D590–D596. doi: 10.1093/nar/gks1219

Romanenko, L. A., Tanaka, N., Svetashev, V. I., Kalinovskaya, N. I., and Mikhailov,V. V. (2015). Rheinheimera japonica sp. nov., a novel bacterium withantimicrobial activity from seashore sediments of the Sea of Japan. Arch.Microbiol. 197, 613–620. doi: 10.1007/s00203-015-1095-2

Saito, M., Seki, M., Iida, K., Nakayama, H., and Yoshida, S. (2007). A novel agarmedium to detect hydrogen peroxide-producing bacteria based on the Prussianblue-forming reaction. Microb. Immunol. 51, 889–892. doi: 10.1111/j.1348-0421.2007.tb03971.x

Schrallhammer, M., Ferrantini, F., Vannini, C., Galati, S., Schweikert, M., Görtz,H. D., et al. (2013). ‘Candidatus Megaira polyxenophila’ gen. nov., sp. nov.:considerations on evolutionary history, host range and shift of early divergentRickettsiae. PLoS One 8:e72581. doi: 10.1371/journal.pone.0072581

Skovorodkin, I. N. (1990). A device for immobilizing biological objects in the lightmicroscope studies. Tsitologiya 32, 301–302.

Sogin, M. L., Swanton, M. T., Gunderson, J. H., and Elwood, H. J. (1986).Sequence of the small subunit ribosomal RNA Gene from the hypotrichousciliate Euplotes aediculatus. J. Protozool. 33, 26–29. doi: 10.1111/j.1550-7408.1986.tb05550.x

Stubbendieck, R. M., and Straight, P. D. (2016). Multifaceted interfaces of bacterialcompetition. J. Bacteriol. 198, 2145–2155. doi: 10.1128/JB.00275-16

Stubbendieck, R. M., Vargas-Bautista, C., and Straight, P. D. (2016). Bacterialcommunities: interactions to scale. Front. Microbiol. 7:1234. doi: 10.3389/fmicb.2016.01234

Sun, S., Dai, X., Sun, J., Bu, X., Weng, C., Li, H., et al. (2016). A diketopiperazinefactor from Rheinheimera aquimaris QSI02 exhibits anti-quorum sensingactivity. Sci. Rep. 6:39637. doi: 10.1038/srep39637

Szokoli, F., Castelli, M., Sabaneyeva, E., Schrallhammer, M., Krenek, S., Doak, T. G.,et al. (2016). Disentangling the taxonomy of Rickettsiales and description of twonovel symbionts (“Candidatus Bealeia paramacronuclearis” and “CandidatusFokinia cryptica”) sharing the cytoplasm of the ciliate protist Parameciumbiaurelia. Appl. Environ. Microbiol. 82, 7236–7247. doi: 10.1128/AEM.02284-16

Vannini, C., Ferrantini, F., Schleifer, K. H., Ludwig, W., Verni, F., and Petroni, G.(2010). “Candidatus Anadelfobacter veles” and “Candidatus Cyrtobactercomes”, two new Rickettsiales species hosted by the protist ciliate Euplotesharpa (Ciliophora, Spirotrichea). Appl. Environ. Microbiol. 76, 4047–4054.doi: 10.1128/AEM.03105-09

Vannini, C., Petroni, G., Verni, F., and Rosati, G. (2005). Polynucleobacter bacteriain the brackish-water species Euplotes harpa (Ciliata, Hypotrichia). J. Eukaryot.Microbiol. 52, 116–122. doi: 10.1111/j.1550-7408.2005.04-3319.x

Vannini, C., Pöckl, M., Petroni, G., Wu, Q. L., Lang, E., Stackebrandt, E.,et al. (2007). Endosymbiosis in statu nascendi: close phylogeneticrelationship between obligately endosymbiotic and obligately free-livingPolynucleobacter strains (Betaproteobacteria). Environ. Microbiol. 9, 347–359.doi: 10.1111/j.1462-2920.2006.01144.x

Vannini, C., Rosati, G., Verni, F., and Petroni, G. (2004). Identification ofthe bacterial endosymbionts of the marine ciliate Euplotes magnicirratus(Ciliophora, Hypotrichia) and proposal of “Candidatus Devosia euplotis”.Int. J. Syst. Evol. Microbiol. 54, 1151–1156. doi: 10.1099/ijs.0.02759-0

Vannini, C., Sigona, C., Hahn, M., Petroni, G., and Fujishima, M. (2017). Highdegree of specificity in the association between symbiotic betaproteobacteriaand the host Euplotes (Ciliophora, Euplotia). Eur. J. Protistol. 59, 124–132.doi: 10.1016/j.ejop.2017.04.003

Wildschutte, H., Wolfe, D. M., Tamewitz, A., and Lawrence, J. G. (2004). Protozoanpredation, diversifying selection, and the evolution of antigenic diversity inSalmonella. Proc. Natl. Acad. Sci. U.S.A. 101, 10644–10649. doi: 10.1073/pnas.0404028101

Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

Copyright © 2019 Chiellini, Pasqualetti, Lanzoni, Fagorzi, Bazzocchi, Fani, Petroniand Modeo. This is an open-access article distributed under the terms of the CreativeCommons Attribution License (CC BY). The use, distribution or reproduction inother forums is permitted, provided the original author(s) and the copyright owner(s)are credited and that the original publication in this journal is cited, in accordancewith accepted academic practice. No use, distribution or reproduction is permittedwhich does not comply with these terms.

Frontiers in Microbiology | www.frontiersin.org 15 March 2019 | Volume 10 | Article 510